1. Technical Field
The present disclosure relates to an imaging method, and more particularly, to an imaging method for acquiring a three-dimensional image of a sample such as a cell using upconverting nanoparticles.
2. Description of the Related Art
The fluorescence microscope is an optical system that is most widely used to observe biological cells. The fluorescence microscope acquires an image of a cell by making an enlarged cell image formed on an image sensor with fluorescent light emitted from a cell dyed with a fluorescent dye through an objective lens. A charge coupled device (CCD) is an implementation of the image sensor. The optical system of the fluorescence microscope has a conventional wide field microscope as a basic structure thereof. Thereby, not only fluorescent light emitted from the cell and light of the image formed on the CCD but also light which is unfocused and thus fails to form an image enters the image capturing device. Thus, it is difficult to acquire an accurate three-dimensional image of the cell. Due to this issue, contrast and resolution of the acquired three-dimensional image of the cell are degraded. In particular, for a fluorescence microscope employing a cell dyed with a fluorescent dye as a sample, the aforementioned issue is more serious than for a typical optical microscope, and it is difficult to correctly observe a fine structure of the cell.
To address this issue, a confocal laser scanning microscope has been introduced. The confocal laser scanning microscope overcomes the above issue by filtering out light fails to form an image, using a pin-hole as a spatial filter. Accordingly, the confocal laser scanning microscope has higher contrast and higher resolution than a typical wide field microscope. Further, the confocal laser scanning microscope can obtain an optically sectioned image of a cell. Such features of the confocal laser scanning microscope are essential conditions for constructing a three-dimensional image of a cell. Presently, the confocal laser scanning microscope implements a three-dimensional image of a cell with high contrast and high resolution, and thus has become an important indispensable cell imaging system in bioengineering research.
However, to create an image using a confocal laser scanning microscope, a laser beam scanning apparatus for scanning a sample with a laser beam is needed, and a system for regenerating an image from an optical signal reaching an optical detector through the pin-hole is also needed. Accordingly, the confocal laser scanning microscope inevitably uses x-y scanning technique in obtaining a sectioned image, and thus it takes a long time for the confocal laser scanning microscope to measure the three-dimensional image due to the limited speed of data acquisition. Thereby, it is difficult to observe change of the sample in real time using the confocal laser scanning microscope.
The description provided above is merely intended to provide further understanding of the present disclosure, and should not be understood as meaning that the present disclosure corresponds to a prior art technology which is well known to those skilled in the art.